PROJECT SUMMARY The proposed research will address challenges in imaging dynamic structures of proteins and protein complexes in solution. Fluorescence microscopy is currently the dominant technique for chemically specific imaging in solution. Although super-resolution techniques have dramatically pushed the resolution limits of fluorescence microscopy, it is still a major challenge to obtain images of single-molecules and molecular complexes with resolutions comparable to electron microscopy and X-ray crystallography. Our proposed research will develop an atomic force microscopy (AFM) platform that harnesses short-lived DNA/DNA interactions to achieve imaging and three-dimensional reconstruction of chemical groups within individual proteins, multi-enzyme complexes, and complexes between proteins, RNA and DNA with near atomic resolution and in physiologically relevant conditions. Enhancing the imaging speed will enable observation of structural transitions. These capabilities are enabled by a series of innovations: (1) the use of short fragments of DNA as imaging labels, wherein the sequence of DNA encodes color information, (2) the use of short-lived, far from equilibrium interactions that boost resolution and chemical specificity, and (3) the use of specially designed T-shaped atomic force microscopy cantilevers that allow detecting short-lived interactions. The resulting microscopy platform will have a broad impact across many biomedical fields by providing structural information about proteins and protein complexes that are difficult to study via the current methods.